Antibodies against platelet alloantigens play an important role in immune mediated disorders. HPA-1 and HPA-5 are the most important platelet alloantigens related to pathological situations. The approximate genotype frequencies in Caucasians for HPA-5 are 79.4% HPA-5aa, 19.4%, HPA-5ab, 1.2% HPA-5bb. Consequently, a considerable proportion of the population is at risk of being immunized by the HPA-5 antigen either after pregnancy or transfusion. The alloantigen containing the differences between the two allelic forms of HPA-5 is located on the glycoprotein Ia (GPIa) which is non-covalently associated with GPIIa. The HPA-5b allele contains an adenine instead of guanine at base 1648, which results in a glutamic acid to lysine amino acid substitution responsible for immunological distinction of the two alleles. GPIa/IIa (VLA-2, CD49b/CD29) is a member of the integrin family of adhesion receptors involved in cell-cell and cell-matrix interactions. Functionally GPIa contains a collagen binding site. If blood vessels become damaged, collagen in the subendothelium is exposed. As an important physiological activator of platelets, the collagen binding to GPIa/IIa leads to the induction of aggregation and adhesion.
Alloantibodies against HPA-5b on GPIa/IIa were first found in cases of neonatal alloimmune thrombocytopenia (NAIT) and in sera from polytransfused patients. In our hands development of NAIT caused by an alloimmunisation against HPA-5b is approximately as frequent as against HPA-1a, although clinical symptoms are often less severe. The incidence of NAIT is 1.3 per 1000 live births. The risk of morbidity is significant: 20% of affected infants have neurologic sequelae and the death rate is about 10%. Most cases of NAIT are caused by immunization of the mother against the platelet-specific alloantigen HPA-1a (80% of NAIT cases), and the remaining cases are caused predominantly by anti-HPA-5b response. Allo-anti-HPA-5b are more frequent than allo-anti-HPA-1a in case of platelet refractoriness. In other studies the frequency of anti HPA-5b alloantibodies is much higher. In the Lyon Blood Center the frequency of anti HPA-5b is 90%. Even low levels of alloantibodies as induced in mild or subclinic cases of NAIT lead to immunological memory, known to increase the risk of posttransfusion purpura (PTP) for years after delivery. The clinical course of PTP is characterized by pronounced thrombocytopenia and severe hemorrhagic diathesis about one week after the responsible transfusion. Serious complications related to platelet alloantigens are not only found in situations of alloincompatibility in pregnancy, but also in polytransfused patients and even in cases of bone marrow transplantation. Taken together, these clinical complications emphasise the need for routine platelet typing in parallel with the routine typing for the AB0 blood group system. For this reason and for detection of platelet alloantibodies, a simple and reliable diagnostic assay, that is suitable for widespread use and not only performed by a few reference laboratories, is required.
Up to now, the phenotyping for HPA-5b has been dependent on the availability of rare polyclonal human sera containing platelet specific alloantibodies. Most of these sera, however, are impaired by the presence of alloantibodies especially against HLA class I antigens and have to be submitted to extensive absorption and purification protocols. Furthermore, the quality of these antisera is subject to high batch-to-batch variation due to the fluctuations of antibody titers in donor sera.
Recently, a specific human alloantibody against HPA-1a on GPIIb/IIIa was isolated by phage display technology and produced as full-length IgG1 antibody. For the selection of anti-HPA-1a specific phages the purified GPIIb/IIIa was available. The IIb/IIIa containing the HPA-1 is expressed at 50 000 to 80 000 copies per platelet, whereas expression of GPIa/IIa with the HPA-5 is 100 fold lower (800 to 2800 copies per platelet). Due to this low expression it was extremely difficult to purify enough GPIa/IIa which could then be used for the selection of anti-HPA-5b specific Fab-phages. It is why this technology is inefficient to produce anti HPA-5b monoclonal antibody.
Further, establishing a platelet typed blood product panel is complicated by current phenotyping assays that require potent polyclonal anti-HPA-5b sera. Also, the Monoclonal Antibody-specific Immobilization of Platelet Antigens (MAIPA) assay, as a standard method for the detection of alloantibodies in serum and plasma and used to phenotype platelets, is dependent on polyclonal antisera. The polyclonal antisera are rare and dependent on the blood donations from women alloimmunized by their babies. Furthermore the use of such sera may be impaired by the presence of antibodies against other polymorphic antigens such as the AB0 blood groups or HLA. Complicated absorption and purification protocols are required before the polyclonal antiserum is suitable for use in diagnostic assays.
Thus, the problem underlying the present invention is to provide new means for detecting human platelet alloantigens
The solution to the above technical problem is achieved by the embodiments characterized in the claims.